In a developing multicellular organism, the formation of the anteroposterior (A-P), dorsoventral (D-V), and left-right (L-R) body axes is a critical early step in the formation of the body plan. Secreted signaling molecules are important participants in the specification of these body axes. One such family of signaling molecules, encoded by the Wnt genes, is known to regulate the development of the A-P axis. Two different classes of Wnts have been identified and signal specificity is thought to arise via selective activation of different signal transduction pathways. One such pathway is known as the "canonical" Wnt/beta-catenin pathway and signals primarily via beta-catenin, while the other "alternative" or Wnt-planar cell polarity (PCP) pathway is less well-defined but likely involves the Rho family of GTPases. The primary focus of our research is to understand the molecular mechanisms by which these different classes of Wnts regulate the specification and patterning of the body axes during early mouse development. The alignment of the L-R body axis relative to the A-P and D-V body axes is central to the organization of the vertebrate body plan. The mouse node/organizer is known to play a key role in the establishment of asymmetry along the L-R axis. We have recently identified Wnt3a as being a critical gene required for formation of the L-R axis. 50% of homozygous Wnt3a mutant embryos display situs inversus or situs ambiguous indicating that the L-R axis is randomized. Wnt3a is expressed just prior to node formation in the primitive streak adjacent to the node and presumably functions to regulate the asymmetric expression of left-determining genes in the node. We are currently taking molecular, genetic, and cell biological approaches to examine the mechanisms underlying the control of L-R asymmetric gene expression by Wnt3a. Wnts and alternative signal transduction pathways in the regulation of cell polarity and morphogenesis. The proper orientation and movement of cells in a three-dimensional framework defined by the body plan is critical for the formation, growth, and elongation of the mammalian embryo. Wnts are known to regulate cell polarity and the orientation of mitotic cells and are thought to do so by signaling through the "alternative" Wnt signaling pathway. Targeted mutations in Wnts thought to signal via these pathways, such as Wnt5a, have demonstrated that Wnt5a is required for proper extension of the A-P body axis as the mutant trunk is shortened and embryos lack tails. Additional defects in the outgrowth of the face, tongue, limbs, external ear, external genitalia, and the gastrointestinal tract are observed. The abnormal morphologies observed in these disparate structures bear a striking resemblance to each other, suggesting that Wnt5a plays a fundamental role in regulating tissue morphogenesis. Using a combination of embryological techniques (e.g., lineage tracing, transplantation, whole embryo electroporation, and culture) coupled with molecular and cellular approaches, our current studies are directed towards understanding how Wnt5a may regulate the cell-cell interactions that lead to tissue polarity and convergent-extension movements during gastrulation. The small GTPase RhoA is known to be a key regulator of cell polarity and morphogenesis and has been recently shown in frogs to be coupled to the Wnt pathway by a formin-homology protein called Daam. In collaboration with Xi He (Harvard Med), we have cloned two mouse homologs of Daam. Interestingly, their expression patterns are similar to that of Wnt5a, consistent with the Daam molecules signaling downstream of Wnt5a in the Wnt-PCP pathway. We are currently taking both gain- and loss-of-function approaches in vitro and in vivo to understand how the Daam proteins control the Wnt/PCP pathway to regulate cell and tissue polarity during mouse embryogenesis.